The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo-and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo-and polypeptides. The application of this approach to protein studies is discussed.

The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one-dimensional nuclear magnetic resonance and used to determine the thermodynamic parameters of global unfolding at mild solution conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by standard denaturation methods, most notably due to the slow cis-trans isomerization of the prolyl peptide bond. This difference can be quantitatively calculated from basic principles. Even with these corrections, HX experiments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than lo3 are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (0H"ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 A. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.